Decreased protein yields after successive passaging?

I am working on a GST fusion protein expression study and need to make several passages of my BL21 stock for later fusion protein expression studies. I began by creating a MCS and freezing this down. From this I can obtain ~43mg/100ml culture after 3hr IPTG induction.

From the MCS, I produced MCS+1, which was produced by growing BL21 at 37C until mid-log phase. At this point, I froze down aliquots. The problem begins when I try performing an expression exp. with MCS+1. Using the same induction protocol as MCS, I can only obtain ~29-31mg/100ml culture. I've repeated this a few times with comparable results. Is this to be expected? I've postulated that perhaps my pGEX plasmid is getting "kicked out" with each passage, but not sure how this is possible under Amp selection. I need to get out to MCS+4 and am concerned my yields will further diminish with each passage. To counteract this, I have begun expression exps using TB media (instead of LB). No results yet, but I assume my yields will be better, but concerned each passage will still be lower. Any thoughts?

My suspect is your stock might been contaminated. I suggest you to use Carbenicillin instead of Amp: more stable to be degraded by beta-lactamase. For prolong storage, amp is more prone to be degraded.

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